Prodrug Integrated Envelope on Probiotics to Enhance Target Therapy for Ulcerative Colitis

Abstract

Ulcerative colitis (UC), affecting millions of patients worldwide, is associated with disorders of the gut microbiota. Probiotics-based therapy positively regulating the community structure of gut microbiota is regarded as an efficient intervention for UC. However, oral probiotics delivery is restricted by limited bioactivity, short retention time, complex pathological condition, and single therapeutic efficacy. Here, a bioengineered probiotic decorated with a multifunctional prodrug coating is constructed to ameliorate the aforementioned shortcomings. The results of UC mice induced by dextran sulfate sodium demonstrate that the intrinsic features of the fabricated coating integrate gut microbes protection, colon-targeted drug release, prolonged drug retention, and inflammation regulation. In parallel, the probiotics Lactobacillus rhamnosus GG (LGG) could regulate the composition of the gut microbiota and improve epithelial barrier function, thereby synergistically ameliorating UC. These results provide ample shreds of evidence of the therapeutic effect on UC, therefore, demonstrate a great promise as the potential therapeutic strategy for UC treatment.

Keywords: lactobacillus rhamnosus GG (LGG); oral delivery; prodrug; surface decoration; ulcerative colitis.

Scheme 1

Schematic illustration of the LBL preparation process and the mechanism of LBL treatment of ulcerative colitis (UC).

Figure 1

Fabrication and characterization of LBL. a) Schematic illustration of synthesis and the structure of LPC‐Bal. b) The fabrication process of LBL. c) 600 MHz ¹H nuclear magnetic resonance spectroscopy (¹H NMR) of LPC‐Bal. d) Representative TEM images and e) CLSM images of the native and decorated LGG. The coating and LGG were stained with FITC‐DSPE‐mPEG₂₀₀₀ and Cy5‐NHS, respectively. f) The x–z and y–z views of the fluorescence images processed by Imaris, demonstrating the colocalization of LGG and the coating.

Figure 2

In vitro resistance of LBL to the simulated harsh environments. a) Growth curves of the native and decorated LGG in MRS culture medium at 37 °C and the OD600 was recorded every 30 min for 12 h by a microplate reader (n = 3). b) Schematic representation of the in vitro resistance of LBL against environmental assaults. Survival of the native LGG and LBL after incubation with c) the simulated gastric fluid (SGF) or d) the simulated intestinal fluid (SIF) at 37 °C. The number of the survived bacteria was quantified by spreading 100 µL of the serially diluted samples of each suspension onto MRS agar plates and incubated at 37 °C for 24 h (n = 5). Flow cytometric analysis of LBL after exposure to e) SGF or f) SIF. The coating was stained with FITC‐DSPE‐mPEG₂₀₀₀. g) CLSM images of LBL after incubation with SGF or SIF for the predetermined time interval. The coating and LGG were stained with FITC‐DSPE‐mPEG₂₀₀₀ and Cy5‐NHS, respectively. h) Representative TEM images of LBL after SGF or SIF treatment for predefined time points. Data were shown as mean ± SD. Unpaired t test (two‐tailed) was performed on (c) and (d), ns (no significance), P < 0.01 (**), or P < 0.001 (***).

Figure 3

The anti‐inflammation potential of the coating. a) The release kinetics of balsalazide (Bal) to 5‐aminosalicylic acid (5‐ASA) under sodium dithionite. b) The cumulative release curve of Bal and LPC‐Bal with or without sodium dithionite (n = 9). c) Schematic diagram of inflammation inhibition effect of 5‐ASA in Caco‐2 cells induced by DSS. d–f) Total RNA was extracted from Caco‐2 cells. The mRNA expression levels of IL‐6, IL‐1β, and TNF‐α were determined by qPCR. g) Representative immunofluorescence staining and h) quantitative analysis for TLR4/NF‐kB signaling pathway‐related protein P‐p65 (n = 5). Data are shown as mean ± SD. Significances were determined by one‐way analysis of variance (ANOVA), followed by post hoc pairwise comparisons with the Tukey honest significant difference P < 0.001 (***).

Figure 4

In vivo treatment of LBL against ulcerative colitis (UC). a) Schematic diagram of the treatment of LBL on UC. b) Representative IVIS images of the GI tract 4 h postadministration of the native and decorated LGG. c) Illustration of the experimental protocol. Mice were given drinking water containing 3% DSS for 7 d to induce colitis followed by administration with LBL for 5 d. d) Representative images of the harvested intestinal tracts. e) Length of the colorectum (n = 5). f–h) Serum levels of IL‐6, IL‐1β, and TNF‐α (n = 5). i–k) Total RNA was extracted from the colon (n = 5). The mRNA expression levels of IL‐6, IL‐1β, and TNF‐α were determined by qPCR. l) Representative images of myeloperoxidase (MPO) immunohistochemistry of the colon. m) The mean integrated optical density (IOD) of MPO, quantified by Image J (n = 15). Data are shown as mean ± SD. Significances were determined by one‐way ANOVA, followed by post hoc pairwise comparisons with the Tukey honest significant difference. P < 0.05 (*), P < 0.01 (**), or P < 0.001 (***).

Figure 5

Improvement of the gut barrier after administration of LBL. a) Representative immunofluorescence staining for zona occludens protein‐1 (ZO‐1) of colonic tissue. b) Representative immunofluorescence staining for occludin of colonic tissue. c) Representative photoimages of alcian blue staining of colonic tissue. d) Representative images of H&E in mouse colon tissue. e,f) The fluorescence intensities of ZO‐1 and occludin were measured by Image J (n = 15). g,h) The number and the area of goblet cells were calculated with Image J (n = 15). Data are shown as mean ± SD. Significances were determined by one‐way ANOVA, followed by post hoc pairwise comparisons with the Tukey honest significant difference. P < 0.05 (*), P < 0.01 (**), or P < 0.001 (***).

Figure 6

Regulation of LBL on the gut microbial community. The V3‐V4 region of the bacterial 16S rRNA was sequenced in fecal samples and comparisons were carried out using alpha and beta diversity. a) Chao1, b) Shannon, c) Inverse‐Simpson, d) principal components analysis (PCA), e) principal coordinate analysis (PCoA), and f) nonmetric multidimensional scaling (NMDS) indices of ASV level. g) Heatmap of the ten most abundant taxa at the family level. h) LEfSe histograms. Bacterial taxa that met the criterion of an LDA score >3.5 were considered biomarker taxa. i) Circos diagram to reflect the distribution proportion of the dominant specie in different groups. Data are shown as mean ± SD (n = 5). Significances were determined by one‐way ANOVA, followed by post hoc pairwise comparisons with the Tukey honest significant difference. P < 0.05 (*), P < 0.01 (**), or P < 0.001 (***).